BACKGROUND: The endoplasmic reticulum (ER) transmembrane chaperones DNAJB12(B12) and DNAJB14(B14) are cofactors that cooperate with cytosolic Heat Shock-70 protein (HSC70) facilitating folding/degradation of nascent membrane proteins and supporting the ER-membrane penetration of viral particles. Here, we assessed structural/functional features of B12/B14 with respect to their regulation by ER stress and their involvement in ER stress-mediated protein reflux. METHODS: We investigated the effect of Unfolded Protein Response(UPR)-eliciting drugs on the expression/regulation of B12-B14 and their roles in ER-to-cytosol translocation of Protein Disulfide Isomerase-A1(PDI). RESULTS: We show that B12 and B14 are similar but do not seem redundant. They share predicted structural features and show high homology of their cytosolic J-domains, while their ER-lumen DUF1977 domains are quite dissimilar. Interactome analysis suggested that B12/B14 associate with different biological processes. UPR activation did not significantly impact on B12 gene expression, while B14 transcripts were up-regulated. Meanwhile, B12 and B14 (33.4 kDa isoform) protein levels were degraded by the proteasome upon acute reductive challenge. Also, B12 degradation was impaired upon sulfenic-acid trapping by dimedone. We originally report that knockdown of B12/B14 and their cytosolic partner SGTA in ER-stressed cells significantly impaired the amount of the ER redox-chaperone PDI in a cytosolic-enriched fraction. Additionally, B12 but not B14 overexpression increased PDI relocalization in non-stressed cells. CONCLUSIONS AND GENERAL SIGNIFICANCE: Our findings reveal that B12/B14 regulation involves thiol redox processes that may impact on their stability and possibly on physiological effects. Furthermore, we provide novel evidence that these proteins are involved in UPR-induced ER protein reflux.
Endoplasmic Reticulum , Molecular Chaperones , Molecular Chaperones/metabolism , Endoplasmic Reticulum/metabolism , Cytosol/metabolism , Proteasome Endopeptidase Complex/metabolism , Oxidation-Reduction
BACKGROUND AND AIMS: Redox signaling is involved in the pathophysiology of aortic aneurysm/dissection. Protein Disulfide Isomerases and its prototype PDIA1 are thiol redox chaperones mainly from endoplasmic reticulum (ER), while PDIA1 cell surface pool redox-regulates thrombosis, cytoskeleton remodeling and integrin activation, which are mechanisms involved in aortic disease. Here we investigate the roles of PDIA1 in aortic dissection. METHODS: Initially, we assessed the outcome of aortic aneurysm/dissection in transgenic PDIA1-overexpressing FVB mice using a model of 28-day exposure to lysyl oxidase inhibitor BAPN plus angiotensin-II infusion. In a second protocol, we assessed the effects of PDIA1 inhibitor isoquercetin (IQ) against aortic dissection in C57BL/6 mice exposed to BAPN for 28 days. RESULTS: Transgenic PDIA1 overexpression associated with ca. 50% (p = 0.022) decrease (vs.wild-type) in mortality due to abdominal aortic rupture and protected against elastic fiber breaks in thoracic aorta. Conversely, exposure of mice to IQ increased thoracic aorta dissection-related mortality rates, from ca. 18%-50% within 28-days (p = 0.019); elastic fiber disruption and collagen deposition were also enhanced. The structurally-related compound diosmetin, which does not inhibit PDI, had negligible effects. In parallel, stretch-tension curves indicated that IQ amplified a ductile-type of biomechanical failure vs. control or BAPN-exposed mice aortas. IQ-induced effects seemed unassociated with nonspecific antioxidant effects or ER stress. In both models, echocardiographic analysis of surviving mice suggested that aortic rupture was dissociated from progressive dilatation. CONCLUSIONS: Our data indicate a protective role of PDIA1 against aortic dissection/rupture and potentially uncovers a novel integrative mechanism coupling redox and biomechanical homeostasis in vascular remodeling.
Redox-related plasma proteins are candidate reporters of protein signatures associated with endothelial structure/function. Thiol-proteins from protein disulfide isomerase (PDI) family are unexplored in this context. Here, we investigate the occurrence and physiological significance of a circulating pool of PDI in healthy humans. We validated an assay for detecting PDI in plasma of healthy individuals. Our results indicate high inter-individual (medianâ¯=â¯330â¯pg/mL) but low intra-individual variability over time and repeated measurements. Remarkably, plasma PDI levels could discriminate between distinct plasma proteome signatures, with PDI-rich (>median) plasma differentially expressing proteins related to cell differentiation, protein processing, housekeeping functions and others, while PDI-poor plasma differentially displayed proteins associated with coagulation, inflammatory responses and immunoactivation. Platelet function was similar among individuals with PDI-rich vs. PDI-poor plasma. Remarkably, such protein signatures closely correlated with endothelial function and phenotype, since cultured endothelial cells incubated with PDI-poor or PDI-rich plasma recapitulated gene expression and secretome patterns in line with their corresponding plasma signatures. Furthermore, such signatures translated into functional responses, with PDI-poor plasma promoting impairment of endothelial adhesion to fibronectin and a disturbed pattern of wound-associated migration and recovery area. Patients with cardiovascular events had lower PDI levels vs. healthy individuals. This is the first study describing PDI levels as reporters of specific plasma proteome signatures directly promoting contrasting endothelial phenotypes and functional responses.
Endothelial Cells/metabolism , Phenotype , Protein Disulfide-Isomerases/blood , Proteome , Proteomics , Adult , Biomarkers , Cell Survival , Cells, Cultured , Enzyme-Linked Immunosorbent Assay , Female , Gene Expression , Healthy Volunteers , Humans , Male , Oxidation-Reduction , Platelet Aggregation , Proteomics/methods , Reproducibility of Results
Protein disulfide isomerases including PDIA1 are implicated in cancer progression, but underlying mechanisms are unclear. PDIA1 is known to support vascular Nox1 NADPH oxidase expression/activation. Since deregulated reactive oxygen species (ROS) production underlies tumor growth, we proposed that PDIA1 is an upstream regulator of tumor-associated ROS. We focused on colorectal cancer (CRC) with distinct KRas activation levels. Analysis of RNAseq databanks and direct validation indicated enhanced PDIA1 expression in CRC with constitutive high (HCT116) vs. moderate (HKE3) and basal (Caco2) Ras activity. PDIA1 supported Nox1-dependent superoxide production in CRC; however, we first reported a dual effect correlated with Ras-level activity: in Caco2 and HKE3 cells, loss-of-function experiments indicate that PDIA1 sustains Nox1-dependent superoxide production, while in HCT116 cells PDIA1 restricted superoxide production, a behavior associated with increased Rac1 expression/activity. Transfection of Rac1G12V active mutant into HKE3 cells induced PDIA1 to become restrictive of Nox1-dependent superoxide, while in HCT116 cells treated with Rac1 inhibitor, PDIA1 became supportive of superoxide. PDIA1 silencing promoted diminished cell proliferation and migration in HKE3, not detectable in HCT116 cells. Screening of cell signaling routes affected by PDIA1 silencing highlighted GSK3ß and Stat3. Also, E-cadherin expression after PDIA1 silencing was decreased in HCT116, consistent with PDIA1 support of epithelial-mesenchymal transition. Thus, Ras overactivation switches the pattern of PDIA1-dependent Rac1/Nox1 regulation, so that Ras-induced PDIA1 bypass can directly activate Rac1. PDIA1 may be a crucial regulator of redox-dependent adaptive processes related to cancer progression.
Colonic Neoplasms/metabolism , NADPH Oxidase 1/metabolism , Procollagen-Proline Dioxygenase/metabolism , Protein Disulfide-Isomerases/metabolism , Proto-Oncogene Proteins p21(ras)/metabolism , Caco-2 Cells , Cell Movement/physiology , Cell Proliferation/physiology , Colonic Neoplasms/enzymology , Colonic Neoplasms/pathology , Glycogen Synthase Kinase 3 beta/metabolism , HCT116 Cells , Humans , Reactive Oxygen Species/metabolism , STAT3 Transcription Factor/metabolism , Signal Transduction , Transfection , rac1 GTP-Binding Protein/metabolism
Redox-related plasma proteins are candidate reporters of protein signatures associated with endothelial struc-ture/function. Thiol-proteins from protein disulfide isomerase (PDI) family are unexplored in this context. Here,we investigate the occurrence and physiological significance of a circulating pool of PDI in healthy humans. Wevalidated an assay for detecting PDI in plasma of healthy individuals. Our results indicate high inter-individual(median = 330 pg/mL) but low intra-individual variability over time and repeated measurements. Remarkably,plasma PDI levels could discriminate between distinct plasma proteome signatures, with PDI-rich (> median)plasma differentially expressing proteins related to cell differentiation, protein processing, housekeeping func-tions and others, while PDI-poor plasma differentially displayed proteins associated with coagulation, in-flammatory responses and immunoactivation. Platelet function was similar among individuals with PDI-rich vs.PDI-poor plasma. Remarkably, such protein signatures closely correlated with endothelial function and phe-notype, since cultured endothelial cells incubated with PDI-poor or PDI-rich plasma recapitulated gene ex-pression and secretome patterns in line with their corresponding plasma signatures. Furthermore, such sig-natures translated into functional responses, with PDI-poor plasma promoting impairment of endothelialadhesion to fibronectin and a disturbed pattern of wound-associated migration and recovery area. Patients withcardiovascular events had lower PDI levels vs. healthy individuals. This is the first study describing PDI levels asreporters of specific plasma proteome signatures directly promoting contrasting endothelial phenotypes andfunctional responses.
Redox-related plasma proteins are candidate reporters of protein signatures associated with endothelial struc-ture/function. Thiol-proteins from protein disulfide isomerase (PDI) family are unexplored in this context. Here,we investigate the occurrence and physiological significance of a circulating pool of PDI in healthy humans. Wevalidated an assay for detecting PDI in plasma of healthy individuals. Our results indicate high inter-individual(median = 330 pg/mL) but low intra-individual variability over time and repeated measurements. Remarkably,plasma PDI levels could discriminate between distinct plasma proteome signatures, with PDI-rich (> median)plasma differentially expressing proteins related to cell differentiation, protein processing, housekeeping func-tions and others, while PDI-poor plasma differentially displayed proteins associated with coagulation, in-flammatory responses and immunoactivation. Platelet function was similar among individuals with PDI-rich vs.PDI-poor plasma. Remarkably, such protein signatures closely correlated with endothelial function and phe-notype, since cultured endothelial cells incubated with PDI-poor or PDI-rich plasma recapitulated gene ex-pression and secretome patterns in line with their corresponding plasma signatures. Furthermore, such sig-natures translated into functional responses, with PDI-poor plasma promoting impairment of endothelialadhesion to fibronectin and a disturbed pattern of wound-associated migration and recovery area. Patients withcardiovascular events had lower PDI levels vs. healthy individuals. This is the first study describing PDI levels asreporters of specific plasma proteome signatures directly promoting contrasting endothelial phenotypes andfunctional responses.
